Therapeutic peptides

ABSTRACT

A linear peptide which is an analog of naturally occurring, biologically active substance P having an active site and a binding site responsible for the binding of the peptide to a receptor on a target cell. The analog has a non-peptide bond instead of a peptide bond between an amino acid residue of the active site and an adjacent amino acid residue, or a synthetic, a β-amino acid, or a α-amino acid residue replacing two amino acid residues of the active site.

BACKGROUND OF THE INVENTION

Applicants hereby request priority under 35 U.S.C. § 120. Thisapplication is a continuation-in-part of Coy et al., U.S. patentapplication Ser. No. 394,727, filed Aug. 16, 1989, abandoned which is acontinuation-in-part of Coy et al., U.S. patent application Ser. No.317,941, filed Mar. 2, 1989, now abandoned, which is acontinuation-in-part of Coy et al., U.S. patent application Ser. No.282,328, filed Dec. 9, 1988, now U.S. Pat. No. 5,162,497, which in turnis a continuation-in-part of Coy et al., U.S. patent application Ser.No. 257,998, filed Oct. 14, 1988, now abandoned, which in turn is acontinuation-in-part of Coy et al., U.S. patent application Ser. No.248,771, filed Sep. 23, 1988, now abandoned, which in turn is acontinuation-in-part of Coy et al., U.S. patent application Ser. No.207,759, filed Jun. 16, 1988, now abandoned, which in turn is acontinuation-in-part of Coy et al., U.S. patent application Ser. No.204,171, filed Jun. 8, 1988, now abandoned, which in turn is acontinuation-in-part of Coy et al., U.S. patent application Ser. No.173,311, filed Mar. 25, 1988, now abandoned, which in turn is acontinuation-in-part of Coy et al., U.S. patent application Ser. No.100,571, filed Sep. 24, 1987, now abandoned.

This invention relates to therapeutic peptides, in particular analogs ofthe naturally occurring peptide substance P.

Substance P (SP) has numerous pharmacologic effects includingvasodilation and hypotension, contraction of non-vascular smooth muscle,stimulation of salivary and pancreatic secretion, depolarization ofvarious neurons and histamine release from mast cells. SP is thought toplay a variety of physiological roles (many of which are associated withthe induction of pain). These include regulation of peristalsis andsmooth muscle activity in the gastrointestinal tract, regulation ofsalivary and pancreatic secretion, regulation of the inflammatoryresponse to peripheral tissue injury, neurotransmission, and regulationof neuro-immunomodulation. Mantyh et al. (1989) Proc. Natl. Acad. Sci.USA 86:5193 reports the presence of substance P receptors at wound sitesin the central nervous system and suggests that SP may be involved inregulating the response to injury in the central nervous system as wellas in peripheral tissues. Substance P is also a proliferative agent,stimulating the proliferation of fibroblasts, T-lymphocytes, endothelialcells, smooth muscle cells and astrocytes.

SP belongs to a family of bioactive peptides known as the tachykinins.The structure, activity, and function of SP and other tachykinins arediscussed in Payan (1989) Ann. Rev. Med. 40:341. As discussed in Payan,SP shares common pharmacological properties and a conserved carboxylterminal sequence (Phe-X-Gly-Leu-Met-NH₂, where X is a branchedaliphatic or aromatic amino acid residue) with the other tachykinins.The principle biological activities, and the ability to bind to areceptor, reside in the carboxyl terminal sequence of these peptides.Selectivity toward a specific tachykinin receptor is determined by theamino-terminal sequence of the peptides Iverson et al., (1989) TheTachykinin System, Abstract presented at the 11th American PeptideSymposium. The conservation of carboxyl terminal sequence extends beyondSP and other mammalian tachykinins to other bioactive peptides, as shownin Table 1.

Numerous derivatives of SP, made by side chain modification and/orD-amino acid substitution, have been shown to act as SP receptorantagonists. Folkers, U.S. Pat. No. 4,481,139, describes substance Pantagonists made by D or L amino acid substitution. These antagonistsinclude the undecapeptide analog spantide(H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Leu-NH₂) as well astruncated analogs of substance P. Jensen et al. (1988) Am. J. ofPhysiol. 254:G883 characterized the ability of various SP antagonists toinhibit the action of bombesin. Jensen et al. studied four SP analogues:H-Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe-Phe-D-Trp-Leu-Met-NH₂ ;H-Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Met-NH₂ ;H-D-Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Leu-NH₂ ; andH-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Leu-NH₂. Jensen et al.also studied two SP analogues with the first 3 amino acid residuesdeleted, H-D-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Met-NH₂ andH-D-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH₂. None of these receptorbinding peptides are, however, specific to the SP receptor. All werefound to inhibit bombesin-stimulated amylase release. Jensen et al.concluded that "the ability to inhibit the action of bombesin is ageneral property of SP analogues that also function as SP receptorantagonists and that the SP receptor antagonists are each inhibiting theaction of bombesin by functioning as bombesin receptor antagonists."

Woll et al. (1988) Proc. Nat. Acad. Sci. USA 85:1857 found the substanceP antagonist H-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Leu-NH₂ apotent bombesin antagonist in murine Swiss 3T3 cells.

Agonists and antagonists of a wide spectrum of biologically activepeptide hormones, including substance P, have been synthesized by theintroduction of modification of the peptide bonds of the peptidehormone, see Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides, and Proteins (B. Weinstein, ed.) M. Dekker, New Yorkand Basel, pp. 267-357, for a recent review of the field.

Abbreviations (uncommon): ##STR1## Lys-ε-NHR =lysine wherein the ε-Natom carries an R group, R being any of H, C₁₋₁₂ alkyl, C₇₋₁₀phenylalkyl, or COE where CO is a carbonyl group and E, attached to C ofCO, is C₁₋₂₀ alkyl, C₃₋₂₀ alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, orC₇₋₁₀ phenylalkyl. (The symbol "COE" or the like is used to denote thesame structure hereinafter.) ##STR2## Sar=sarcosine Sta (statine)=

(3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid, and has the chemicalstructure: ##STR3## AHPPA= (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoicacid, and has the chemical structure: ##STR4## ACHPA= (3S,4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid and has the chemicalstructure: ##STR5## Cpa=Chloro-PhenylalanineSP=H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂ (substance P).

SUMMARY OF THE INVENTION

In general, the invention features a linear (i.e., non-cyclic) peptidewhich is an analog of a naturally occurring, biologically active peptidehaving an active site and a binding site responsible for the binding ofthe peptide to a receptor on a target cell; cleavage of a peptide bondin the active site of the naturally occurring peptide is unnecessary forin vivo biological activity. The analog has one of the followingmodifications: (a) a non-peptide bond instead of a peptide bond betweenan amino acid residue of the active site and an adjacent amino acidresidue, (b) a replacement of two amino acid residues within the activesite with a synthetic amino acid residue, e.g., statine, AHPPA, ACHPA, aβ-amino acid residue, or a γ-amino acid residue, (c) a deletion of anamino acid residue within the active site and a modification of an aminoacid residue outside of the active site, or (d) the presence of anN-terminal amino acid residue that is not the naturally occurring aminoacid residue of said naturally occurring, biologically active peptide(where β- or γ- is not designated an amino acid is an α-amino acid).

In preferred embodiments the analog is capable of acting as acompetitive inhibitor of the naturally occurring peptide by binding tothe receptor and, by virtue of one of the modifications, failing toexhibit the in vivo biological activity of the naturally occurringpeptide.

In preferred embodiments the active site of the linear peptide is in thecarboxyl terminal-half of the linear peptide. The linear peptide has oneof the following modifications: (a) a non-peptide bond instead of apeptide bond between an amino acid residue of the active site and anadjacent amino acid residue, (b) a replacement of two amino acidresidues within the active site with a synthetic amino acid residue,e.g., statine, AHPPA, ACHPA, a β-amino acid residue, or a γ-amino acidresidue, (c) a deletion of an amino acid residue within the active siteand a modification of an amino acid residue outside of the active site,or (d) the presence of an N-terminal amino acid residue that is not thenaturally occurring amino acid residue of said naturally occurring,biologically active peptide.

In preferred embodiments the active site of the linear peptide is in thecarboxyl terminal-half of the linear peptide. The linear peptide has oneof the following modifications, (a) a non-peptide bond instead of apeptide bond between the carboxyl terminal amino acid residue and theadjacent amino acid residue, or (b) a statine or AHPPA or ACHPA, β-aminoacid, or γ-amino acid residue in place of the naturally occurringcarboxyl terminal and adjacent amino acid residues.

In preferred embodiments the linear peptide is an analog of naturallyoccurring biologically active substance P. The analog of substance P hasone of the following modifications, (a) a non-peptide bond instead of apeptide bond between the carboxyl terminal amino acid residue and theadjacent amino acid residue, or (b) a statine or AHPPA or ACHPA, β-aminoacid, or γ-amino acid residue in place of the naturally occurringcarboxyl terminal and adjacent amino acid residues.

In preferred embodiments the analog of substance P is capable of actingas a competitive inhibitor of the naturally occurring substance P bybinding to the receptor and, by virtue of one of the modifications,failing to exhibit the in vivo biological activity of the naturallyoccurring peptide.

In preferred embodiments the linear peptide is an analog of substance Pwith an active site in the carboxyl terminal-half of the linear peptide.The analog of substance P has one of the following modifications: (a) anon-peptide bond instead of a peptide bond between the carboxyl terminalamino acid residue and the adjacent amino acid residue, or (b) a statineor AHPPA or ACHPA or β-amino acid or γ-amino acid residue in place ofthe naturally occurring carboxyl terminal and adjacent amino acidresidues.

The linear peptides for which introduction of a non-peptide bond betweentwo amino acid residues, or the replacement of two natural amino acidresidues with a synthetic amino acid residue, a β-amino acid residue, ora γ-amino acid residue, or the deletion ("des") of the C-terminal aminoacid residue useful in creating or enhancing antagonist activity arethose in which activity is associated with the two C-terminal amino acidresidues of the amino acid chain. Therefore, the active site of thenaturally occurring peptide of which the peptides of the invention areanalogs preferably includes at least on amino acid residue in thecarboxy terminal half of the peptide, and the linear peptide of theinvention includes that amino acid residue in its carboxy terminal half.Modifications can be introduced in a region involved in receptorbinding, or in a non-binding region.

By non-peptide bond is meant that the carbon atom participating in thebond between two residues is reduced from a carbonyl carbon to amethylene carbon, i.e., CH₂ --NH; or CH₂ --S, CH₂ --O, CH₂ --CH₂, CH₂--CO, or CO--CH₂. (A detailed discussion of the chemistry of non-peptidebonds is given in Coy et al. (1988) Tetrahedron 44,3:835-841, herebyincorporated by reference, Tourwe (1985) Janssen Chim. Acta 3:3-15,17-18, hereby incorporated by reference, and Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides, and Proteins, (B. Weinstein,ed.) M. Dekker, New York and Basel, pp. 267-357, hereby incorporated byreference.)

Preferably, analogs of the invention are 25% homologous, mostpreferably, 50% homologous, with the naturally occurring peptides.

One modification of the naturally occurring peptide to create anantagonist is to employ, as the amino terminal residue, an aromaticD-isomer of an amino acid, or an alkylated amino acid. (Where "D" is notdesignated as the configuration of an amino acid, L is intended.)

One class of peptide of the invention includes the substance P analogsof the formula: ##STR6## wherein A¹ =the D or L isomer of Arg, Lys, orLys-ε-NH-R₂₀ in which R₂₀ is any of H, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl,C₁₋₁₂ acyl, or COE₁₀ with E₁₀ being C₁₋₂₀ alkyl, C₃₋₂₀ alkenyl, C₃₋₂₀alkynyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl; or is deleted; the D orL isomer Pro; or is deleted;

A³ =the D or L isomer of Lys, or Lys-ε-NH-R₂₂ in which R₂₂ is any of H,C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, C₁₋₁₂ acyl, or COE₁₂ with E₁₂ beingC₁₋₂₀ alkyl, C₃₋₂₀ alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀phenylalkyl; or is deleted;

A⁴ =the D or L isomer of Pro; or is deleted;

A⁵ =the D or L isomer of Asp, Gln, β-Nal, Trp, Phe, o-X-Phe in which Xis F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂,OH, or CH₃ ; or is deleted;

A⁶ =the D or L isomer of Ala, Arg, Ser, Pro, Gln, pGlu, Asn, β-Nal, Trp,Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe inwhich X is F, Cl, Br, NO₂, OH, or CH₃ ;

A⁷ =the D or L isomer of Val, Thr, Phe, Trp, βNal, o-X-Phe in which X isF, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH,or CH₃ ;

A⁸ =the identifying group of Gly or the D or L isomer of Val, Trp,β-Nal, Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phein which X is F, Cl, Br, NO₂, OH, or CH₃ ;

A⁹ =Gly or the D or L isomer of Sar, His, Trp, β-Nal, Phe, o-X-Phe inwhich X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl,Br, NO₂, OH, or CH₃ ;

A¹⁰ =Gly or the identifying group of the D or L isomer of Trp, β-Nal,Leu, Nle, Ala, cyclohexyl-Ala, Val, Ile, Met, Phe, o-X-Phe in which Xis, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂,OH, or CH₃ ;

A¹¹ =Gly or the identifying group of the D or L isomer of Trp, β-Nal,Leu, Nle, Ala, Val, Ile, Met, Phe, o-X-Phe in which X is F, Cl, Br, NO₂,OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ; or isdeleted;

R₅ is any one of CH₂ -NH, CH₂ -S, CH₂ -O, CH₂ -CH₂, CH₂ -CO, or CO--CH₂; R₆ is C; and V¹ is ##STR7## in which each R₁₀, R₁₁, and R₁₂independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, or C₁₂₋₂₀naphthylalkyl; further provided that, where A⁵ is Asp, A is Ser, A⁶ isPhe, A⁸ is Val, A⁹ is Gly, A¹⁰, A¹¹ is Leu is Leu, and R₅ is CH₂ NH, atleast one of A¹, A², A³, or A⁴, must be present; or said analog with oneor both of the hydrogens of its α-amino group at the N-terminus replacedby one or both of ^(R) 1 and R₂, R₁, and R₂, independently, being C₁₋₁₂alkyl, C₇₋₁₀ phenylalkyl, or COE₁₄ with E₁₄ being C₁₋₂₀ alkyl, C₃₋₂₀alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl, providedthat when one of R₁ or R₂ is COE₁₄ .sub., only one of said hydrogens isreplaced by R₁ or R₂ ; or a pharmaceutically acceptable salt thereof.

Note that amino acid residues, such as Ser, Arg, Leu, and the like,stand for the structure NH-CH(I)-CO, where I is the identifying group ofan amino acid. For example, I is CH₂ OH for Ser. However, forpyroglutamate, see below.

Another class of peptide of the invention includes the substance Panalogs of the formula: ##STR8## wherein A²¹ =the D or L isomer of Arg,Lys, or Lys-ε-NH-R₈₀ in which R₈₀ is any of H, C₁₋₁₂ alkyl, C₇₋₁₀phenylalkyl, C₁ -C₁₂ acyl, or COE₂₀ with E₂₀ being C₁₋₂₀ alkyl, C₃₋₂₀alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl; or isdeleted;

A²² =the D or L isomer of Pro; or is deleted;

A²³ =the D or L isomer of Lys or Lys-ε-NH-R₈₂ in which R₈₂ is any of H,C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, C₁ -C₁₂ acyl, or COE₂₂ with E₂₂ beingC₁₋₂₀ alkyl, C₃₋₂₀ alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀phenylalkyl; or is deleted;

A²⁴ =the D or L isomer of Pro; or is deleted;

A²⁵ =the identifying group of the D or L isomer of Asp, Gln, β-Nal, Trp,Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe inwhich X is F, Cl, Br, NO₂, OH, or CH₃ ; or is deleted together withNH--CH--R₅₄ bonded thereto;

A²⁶ =the identifying group of the D or L isomer of Arg, Sar, Pro, Gln,pGlu, Phe, Trp, cyclohexyl-Ala, or Asn;

A²⁷ =the identifying group of D-Trp; or the identifying group of the Dor L isomer of Leu, Phe, or cyclohexyl-Ala;

A²⁸ =the identifying group of the D or L isomer of any one of the aminoacids Val, β-Nal, Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃,or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ;

A²⁹ =the identifying group of the amino acid D-Trp; or the identifyinggroup of the D or L isomer of any of Leu, Phe, or cyclohexyl-Ala;

A³⁰ =the identifying group of the D or L isomer of any one of the aminoacids Leu, Nle, Ala, cyclohexyl-Ala, Ala, Val, Ile, Met, Gly, Phe, Trp,β-Nal, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe inwhich X is F, Cl, Br, NO₂, OH, or CH₃ ;

A³¹ =the identifying group of the D or L isomer of any one of the aminoacids Trp, β-Nal, Leu, Nle, Ala, Val, Ile, Met, Gly, Phe, o-X-Phe inwhich X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl,Br, NO₂, OH, or CH₃ ;

R₅₄ is CO--NH or CO--NCH₃ if A²⁵ with NH--CH--R₅₄ is not deleted, is NHif A²⁵ with NH--CH--R₅₄ is deleted; each R₅₆, R₆₂, and R₆₄,independently, is any of CO--NH, CH₂ --NH, CH₂ --S, CH₂ --O, CH₂CH_(2--CH) ₂, CH₂ --CO, or CO--CH₂ ; R₅₈ is CO--NR₆₉ in which R₆₉ is Hor C₁₋₁₂ alkyl, CH₂ --NH, CH₂ --S, CH₂ --O, CH₂ --CH₂, CH₂ --CO, orCO--CH₂ ; R₆₀ is CO--NH; and V¹⁰ is ##STR9## in which each R₆₆, R₆₇, andR₆₈ independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, or C₁₂₋₂₀naphthylalkyl; provided that, at least one of R₅₆, R₅₈, R₆₂, or R₆₄ isother than either CO--NH or CO--NR₆₉ ; or said analog with one or bothof the hydrogens of its α-amino group at the N-terminus replaced by oneor both of R₅₁ and R₅₂, R₅₁ and R₅₂, independently, being C₁₋₁₂ alkyl,C₇₋₁₀ phenylalkyl, or COE₂₄ with E₂₄ being C₁ -C₂₀ alkyl, C₃₋₂₀ alkenyl,C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl, provided thatwhen one of R₅₁ or R₅₂ is COE₂₄, only one of said hydrogens is replacedby R₅₁ or R₅₂ ; or a pharmaceutically acceptable salt thereof.

Examples of preferred peptides are:

H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leuψ[CH2--NH]Leu-NH₂ ;H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trpψ[CH₂ --NH]Leu-Nle-NH₂ ;H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trpψ[CH₂ --NH]Phe-D-Trp-Leu-Nle-NH₂ ;H-D-Arg-Pro-Lys-Pro-Gln-Glnψ[CH₂ --NH]D-Trp-Phe-D-Trp-Leu-Nle-NH₂ orH-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leuψ[CH₂ --NH]Nle-NH₂.(Non-peptide bonds in which the peptide bond are symbolized herein by"ψ[CH₂ --NH]", "Ψ[CH₂ O]" or the like, depending on the types ofmodification on the peptide bond].

In another aspect the invention features a substance Pagonist/antagonist of the formula:

H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leuψ[CH₂ --NH]Leu-NH₂ ;H-Arg-Pro-Lys-Pro-Gln-Gln-Pheψ[CH₂ --NH]Phe-Gly-Leu-Leu-NH₂ ;H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leuψ[CH₂ --O]Leu-NH₂ ;H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Glyψ[CH2--NH]Leu-Leu-NH₂ ;H-D-Phe-Gln-Phe-Phe-Gly-Leuψ[CH₂ --O]Leu-NH₂ ;H-D-Phe-Gln-Phe-Gly-Leuψ[CH₂ --NH]Phe-NH₂ ;H-D-Nal-Gln-Phe-Phe-Gly-Leuψ[CH₂ --NH]Phe-NH₂ ;H-D-Cpa-Gln-Phe-Phe-Gly-Leuψ[CH₂ --NH]Phe-NH₂ ; orH-D-Phe-Phe-Gly-Leuψ[CH₂ -NH]Leu-NH₂.

All these analogs have been shown to possess agonist or antagonistactivity on biosystems such as the guinea pig ileum and/or the ratduodenum.

SP antagonists of the invention are useful in the treatment of a patientsuffering from diseases involving neurogenic inflammation e.g.,rheumatoid arthritis, ulcerative colitis, eczema, and Crohn's disease.The SP antagonists of the invention are useful as antiproliferativeagents e.g., in the treatment of small cell lung carcinoma or disordersinvolving the proliferation of fibroblasts. The antiproliferativeproperties of the SP antagonists of the invention also allow their usein the prevention of glial scarring (thus facilitating nerveregeneration). The action of the antagonists of the invention onneurotransmission allow their use as nonopiate analgesics. Their use asnonopiate analgesics can permit the restoration of opiate response. Theantagonists of the invention are also useful as antisecretory agents,acting e.g., on the salivary glands or on the pancreas.

In the generic formulas given above, when any of R₁, R₂, R₇ -R₁₃, R₅₁,R₅₂, R_(66-R) ₆₈, or R_(70-R) ₇₂ is an aromatic, lipophilic group, thein vivo activity can be long lasting, and delivery of the compounds ofthe invention to the target tissue can be facilitated.

The identifying group of an α-amino acid (for case of pyroglutamate, seebelow) is the atom or group of atoms, other than the α-carbonyl carbonatom, the α-amino nitrogen atom, or the H atom, bound to the asymmetricα-carbon atom. To illustrate by examples, the identifying group ofalanine is CH₃, the identifying group of valine is (CH₃)₂ CH, theidentifying group of lysine is H₃ N⁺ (CH₂)₄ and the identifying group ofphenylalanine is (C₆ H₆)CH₂. The identifying group of a β- or 7-aminoacid is the analogous atom or group of atoms bound to respectively, theβ- or the γ-carbon atom. Where the identifying group of an amino acid isnot specified, it may be a α, β, or γ. In the case of pyroglutamate theidentifying group consists of --NH--CO--CH₂ --CH₂ --.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We first briefly describe the drawings. Drawings

FIG. 1 is a pair of graphs illustrating the effect of pseudopeptides ofspantide, H-D-Arg-Pro-Lys-Printer 08 (HP IID- 32 - Bay)P08.PRSPRSIpanel) on SP- stimulated amylase release from pancreatic acini.

FIG. 2 is a graph showing the effect ofH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leuψ[CH₂ -NH]Leu-NH₂ onSP-stimulated amylase release from pancreatic ancini.

FIG. 3 is a pair of graphs illustrating the ability of various SP andspantide pseudopeptides to inhibit binding of ¹²⁵ I-BH-substance P topancreatic ancini.

FIG. 4 is a graph showing the ability of SP and spantide pseudopeptidesto inhibit binding of ¹²⁵ I-[Tyr⁴ ] bombesin to pancreatic acini.

We now describe the structure, synthesis, and use of preferredembodiments of the invention.

Structure

The peptides of the invention all have modifications, e.g., anon-peptide bond in at least one of the indicated positions in which thecarbon atom participating in the bond between two residues is reducedfrom a carbonyl carbon to a methylene carbon. The peptide bond reductionmethod which yields this non-peptide bond is described in Coy et al.,U.S. patent application, Ser. No. 879,348, now U.S. Pat. No. 4,803,261,assigned to the same assignee as the present application, herebyincorporated by reference. The peptides of the invention can be providedin the form of pharmaceutically acceptable salts. Examples of preferredsalts are those with therapeutically acceptable organic acids, e.g.,acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic,salicylic, methanesulfonic, toluenesulfonic, or pamoic acid, as well aspolymeric acids such as tannic acid or carboxymethyl cellulose, andsalts with inorganic acids such as the hydrohalic acids, e.g.,hydrochloric acid, sulfuric acid, or phosphoric acid.

Synthesis of Substance P Analogs

Solid phase syntheses of peptides, including introduction of reducedpeptide bond, were carried out by the automated methods recentlydescribed by Coy et al. J. Biol. Chem. 263:5056 (1988) and Sasaki et al.Peptides 8:119 (1987) employing Advanced ChemTech ACT 200 machines. Botharticles are incorporated herein by reference.

The synthesis of the substance P antagonistH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leuψ[CH₂ NH]Leu-NH₂ follows. Othersubstance P analogs of the invention can be prepared by making theappropriate modifications of the following synthetic method. Suchmodifications are well known in the art.

The first step is the preparation of the intermediateH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leuψ[CH₂ NH]Leu-benzhydrylamineresin, as follows.

Benzhydrylamine-polystyrene resin (Vega Biochemicals, Inc.) (0.97 g, 0.5mmole) in the chloride ion form is placed in the reaction vessel of aBECKMAN 990B peptide synthesizer programmed to perform the followingreaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid(TFA) in methylene chloride (2 times for 1 and 25 min. each); (c)methylene chloride; (d) ethanol; (e) methylene chloride; and (f) 10%triethylamine in chloroform.

The neutralized resin is stirred withalpha-t-butoxycarbonyl(Boc)-leucine and diisopropylcarbodiimide (1.5mmole each) in methylene chloride for 1 hour, and the resulting aminoacid resin is then cycled through steps (a) to (f) in the above washprogram. Boc-leucine aldehyde (1.25 mmoles), prepared by the method ofFehrentz and Castro, Synthesis, p. 676 (1983), is dissolved in 5 ml ofdry dimethylformamide (DMF) and added to the resin TFA salt suspensionfollowed by the addition of 100 mg (2 mmoles) of sodium cyanoborohydride(Sasaki and Coy, Peptides 8:119-121 (1987); Coy et al., id.) Afterstirring for 1 hour, the resin mixture is found to be negative toninhydrin reaction (1 min.), indicating complete derivatization of thefree amino group.

The following amino acids (1.5 mmole) are then coupled successively inthe presence diisopropylcarbodiimide (1.5 mmole), and the resultingamino acid resin is cycled through washing/deblocking steps (a) to (f)in the same procedure as above: Boc-Gly (Boc-Gly is coupled as a 6Mexcess of the p-nitrophenylester), Boc-Phe, Boc-Phe, Boc-Gln, Boc-Gln,(Boc-Gln is coupled as a 6M excess of the p-nitrophenylester), Boc-Pro,Boc-Lys, Boc-Pro, and Boc-Arg. The completed resin is then washed withmethanol and air dried.

The resin described above (1.6 g, 0.5 mmole) is mixed with anisole (5ml) and anhydrous hydrogen fluoride (35 ml) at 0° C. and stirred for 45min. Excess hydrogen fluoride is evaporated rapidly under a stream ofdry nitrogen, and free peptide is precipitated and washed with ether.The crude peptide is dissolved in a minimum volume of 2M acetic acid andpurified on a column (2.5×90 cm) of SEPHADEX G-25 which is eluted with2M acetic acid, followed by preparative medium pressure chromatographyon a column (1.5×45 cm) of VYDAC C₁₈ silica (10-15 μm) which is elutedwith linear gradients of acetonitrile in 0.1% trifluoroacetic acid usingan ELDEX CHROMATROL GRADIENT CONTROLLER (flow rate 1 ml/min). Wherenecessary, analogues are further purified by re-chromatography on thesame column with slight modifications to the gradient conditions whennecessary. Homogeneity of the peptides was assessed by thin layerchromatography and analytical reverse-phase high pressure liquidchromatography, and purity was 97% or higher. Amino acid analysis gavethe expected amino acid ratios. The presence of the reduced peptide bondwas demonstrated by fast atom bombardment mass spectrometry. Each of theanalogues gave good recovery of the molecular ion corresponding to thecalculated molecular mass.

A statine, AHPPA, ACHPA, β-amino acid, or γ-amino acid residue is addedin the same way as is a natural α-amino acid residue, by coupling as aBoc-amino acid. Statine or Boc-statine can be synthesized according tothe method of Rich et al., 1978, J. Org. Chem. 43; 3624; Rich et al.(1988) J. Org. Chem. 53:869; and Rich et al., 1980, J. Med. Chem. 23:27.AHPPA can be synthesized according to the method of Hui et al., 1987, J.Med. Chem. 30:1287. ACHPA can be synthesized according to the method ofSchuda et al., 1988, Journal of Organic Chemistry 53:873. Boc-coupledsynthetic amino acids are available from Nova Biochemicals(Switzerland), Bachem (Torrance, Calif.), and CalBiochem (San Diego,Calif.).

Peptides containing the CH₂ O peptide bond replacement were synthesizedvia carbodiimide/1-hydroxybenzotriazole-mediated incorporation of theBoc-Leu-Ψ(CH₂ O)-Leu-OH pseudodipeptide unit which was purchased fromNeosystem Laboratories, Strasbourg, France. The crude hydrogenfluoride-cleaved peptides were purified on a SEPHADEX G-25 column,eluted with 2M acetic acid, followed by preparative medium pressurechromatography on a VYDAC C18 silica column (10-15 μm), eluted withlinear gradients of acetonitrile in 0.1% trifluoroacetic acid. Whennecessary, peptides were further purified by re-chromatography on thesame column. Homogeneity of the peptides was assessed by thin layerchromatography and analytical reverse-phase high pressure liquidchromatography. The purity of peptides was at least 97%. The presence ofthe pseudopeptide bonds was demonstrated by fast atom bombardment massspectrometry.

Other compounds can be prepared as above and tested for effectiveness asagonists or antagonists in the following test program.

Phase 1- Amylase Release From Pancreatic Acini

SP stimulates the release of amylase in pancreatic acini. Thestimulation or inhibition of release of amylase by pancreatic acini isused as a measure of, respectively, the agonist or antagonist activityof a peptide. Dispersed acini from the pancreas of one animal aresuspended in 150 ml of standard incubation solution. Amylase release ismeasured as described previously (Gardner et al. (1977) J. Physiol.270:439). Amylase activity is determined by the methods of Ceska et al.(Ceska et al. (1969) Clin. Chim. Acta. 26:437 and Ceska et al. (1969)Clin. Chim. Acta. 26:445) using the PHADEBAS REAGENT. Amylase release iscalculated as the percentage of the amylase activity in the acini at thebeginning of the incubation that was released into the extracellularmedium during the incubation.

Phase 2 - Competitive Inhibition of ¹²⁵ I-Bolton-Hunter-SP Binding

Binding of ¹²⁵ I-Bolton-Hunter-SP (¹²⁵ I-BH-SP) to dispersed pancreaticacini is measured as described previously (Jensen et al. (1984) Biochem.Biophys. Acta. 804:181 and Jensen et al. (1988) Am. J. Physiol.254:G883). Incubations contain 0.125 nM ¹²⁵ I-BH-SP and 0.1% bacitracinin standard incubation buffer and were for 30 min at 37° C. Nonsaturablebinding of 125I-BH-SP is the amount of radioactivity associated with theacini when the incubation contains 0.125 nM ¹²⁵ I-BH-SP plus 1 μMunlabeled SP. All values given are for saturable binding, i.e., bindingmeasured with ¹²⁵ I-BH-SP alone (total binding). In all experimentsnonsaturable binding was<30% of total binding.

Phase 3 - Competitive Inhibition of ¹²⁵ I-[Tyr⁴ ] Bombesin Binding

¹²⁵ I-[Tyr⁴ ]0 Bombesin (2200 Ci/mmol) is prepared using a modificationof the method described previously (Jensen et al. (1978) Proc. Natl.Acad. Sci. USA 75:6139). IODO-GEN (1 mg) is dissolved in 5 ml ofchloroform and 5 μl of this solution (1 μg IODO-GEN) is transferred to avial under a stream of nitrogen. To this vial 50 μl of KH₂ PO₄ (pH 7.4)6 μg [Tyr]bombesin in 5 μl water and 1 mCi , Na¹²⁵ I is added mixed andincubated for 6 min at 4° C. at which time the iodination mixture isadded to a vial of 1M dithiothreitol and incubated at 80° C. for 60 min.The iodination mixture is then loaded onto a SEP-PAK CARTRIDGE andeluted with 0.25M tetraethylammonium phosphate (TEAP) followed by 50%(vol/vol) acetonitrile-0.25M TEAP. ¹²⁵ I-[Tyr⁴ ]bombesin is purifiedusing reverse-phase high-performance liquid chromatography (HPLC) andeluted.

Results of Assays of Test Peptides

A number of analogues of substance P, or of the substance P antagonistspantide, each containing a non-peptide bond, can be synthesized andtested in one or more of the assays described in Phase 114 Phase 3above. The structure of substance P isH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met- NH₂. Spantide is ananalog of SP. The structure of spantide isH-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Leu-NH₂. Stimulation orinhibition of the release of amylase from dispersed pancreatic acini wasused as an assay for SP agonist activity or SP antagonist activityrespectively. At a concentration of 10 μM, 9 of 10 SP and spantidederived pseudopeptides failed to stimulate amylase release when presentalone (Table 2). One peptide, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyΨ[CH₂--NH]Leu-Leu-NH₂, (10 μM) had agonist activity causing a 3-fold increasein amylase release (Table 2). Each of the 9 pseudopeptides withoutagonist activity was examined for activity as a SP antagonist. At aconcentration of 10μM each of the spantide derived pseudopeptideanalogues inhibited 1 nM-SP-stimulated amylase release. (Table 2) ThreeSP pseudopeptides, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂--NH]Leu-NH₂, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-PheΨ[CH₂--NH]Gly-Leu-Leu-NH2, and H-Arg-Pro-Lys-Pro-Gln-GlnΨ[CH₂-NH]Phe-Phe-Gly-Leu-Leu-NH₂ caused inhibition (Table 2).

The relative abilities of each peptide to inhibit SP-stimulated amylaserelease was determined by the effect of peptide dose on inhibition.Dose-inhibition studies were carried out for each of the 9pseudopeptides using a concentration of SP (1 nM) that causeshalf-maximal stimulation. (FIG. 1). Results for the 5 spantide derivedpseudopeptides are shown in FIG. 1, left panel.H-D-Arg-Pro-Lys-Pro-Gln-Gln- H-D-Trp-Phe-D-Trp-LeuΨ[CH₂ --NH]Nle-NH₂ wasequipotent to spantide, causing detectable inhibition at 0.03 μM andhalf-maximal inhibition at 1.8 μM (Table 3).H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-TrpΨ[CH₂ --NH]Leu-Nle-NH₂ was2-fold less potent (IC₅₀ 3.5 μM, Table 3) than spantide.H-D-Arg-Pro-Lys-Pro-Gln-GlnΨ[CH₂ --NH]D-Trp-Phe-D-Trp-Leu-Nle-NH₂ was2.6-times (IC50 4.7 μM) less potent than spantide.H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-TrpΨ[CH₂ ---H]Phe-D-Trp-Leu-Nle-NH₂ was3.5-times (IC₅₀, 6.4-μM) less potent andH-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-PheΨ[CH₂ --NH]D-Trp-Leu-Nle-NH₂ was17-times (IC50, 30 μM) less potent than spantide (Table 3).

Results for the SP pseudopeptide analogues are shown in FIG. 1, rightpanel. H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- LeuΨ[CH₂ --NH]Leu-NH₂ wasthe most potent SP derivative causing detectible inhibition at 0.3 μMand half-maximal inhibition at 7.1 μM (Table 3, right).H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-PheΨ[CH₂ --NH]Gly-Leu- Leu-NH₂ andH-Arg-Pro-Lys-Pro-Gln-GlnΨ[CH₂ --NH]Phe-Phe-Gly-Leu-Leu-NH₂ were lesseffective, causing detectable inhibition at 10 μM. They are,respectively, 7-fold and 46-fold less potent thanH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe- Gly-LeuΨ[CH2--NH]Leu-NH₂ (Table 3,right). H-Arg-Pro-Lys-Pro-Gln-Gln-PheΨ[CH₂ -NH]Phe-Gly-Leu-Leu-NH₂exhibited no inhibitory activity at concentrations as high as 30 μM(FIG. 1, right). The most potent SP derived pseudopeptide antagonist wasH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ --NH]Leu-NH₂ (FIG. 1,Table 2).

The inhibitory effects of the most potent SP derived pseudopeptideantagonist, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly- LeuΨ[CH₂ --NH]Leu-NH₂,are shown in FIG. 2. Acini were incubated with increasing concentrationsof SP. Amylase release was detectible with 0.1 nM SP, was half-maximalwith 1 nM SP, and was maximal with 10 nM (FIG. 2). Addition ofH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ -NH]Leu-NH₂ caused aparallel rightward shift in the dose-response curve for SP-stimulatedamylase release. The shift was proportional to the concentration ofH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ --NH]Leu-NH₂ added butthere was no change in the maximal response (FIG. 2).H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-LeuΨ[CH₂ -NH]Nle-NH₂ gavesimilar results (data not shown).

The interaction of SP and spantide derived analogues with the SPreceptors of pancreatic acini was measured by the ability of a peptideto inhibit the binding of ¹²⁵ I-BH-SP to acini. (See Table 3 and FIG.3.)

The spantide derived pseudopeptides show a range of potencies.H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-LeuΨ [CH₂ NH]Nle-NH₂ isroughly equivalent in potency to spantide causing detectible inhibitionat 0.03 μM and half-maximal inhibition at 2.2 μM (FIG. 3, left, Table3). H-D-Arg-Pro-Lys- Pro-Gln-Gln-D-Trp-Phe-D-TrpΨ[CH₂ --NH]Leu-Nle-NH₂and H-D-Arg-Pro-Lys-Pro-Gln-GlnΨ[CH₂ --NH]D-Trp-Phe-D-Trp-Leu-Nle-NH₂are 2-fold less potent than spantide.H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-TrpΨ[CH₂ --NH]Phe-D-Trp-Leu-Nle-NH₂ was3-times (Ki, 6.3 μM) less potent than spantide.H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-PheΨ[CH₂ --NH]D-Trp-Leu-Nle-NH₂ was7-times (Ki, 14.7 μM) less potent than spantide (FIG. 3, Table 3).

The SP derived pseudopeptides also show a range of potencies.H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ --H]Leu-NH₂ causesdetectable inhibition at 0.1 μM, and half-maximal inhibition at 3 μM.H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyΨ[CH₂ --NH]Leu-Leu-NH₂ is 1.5-foldlower in potency, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-PheΨ[CH₂--NH]Gly-Leu-Leu-NH₂ is 20-times less potent, andH-Arg-Pro-Lys-Pro-Gln-Gln-PheΨ[CH₂ --NH]Phe-Gly-Leu-Leu-NH₂ andH-Arg-Pro-Lys-Pro-Gln-GlnΨ[CH₂ --NH]Phe-Phe-Gly-Leu-Leu-NH₂ are morethan 62-times less potent than H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ --NH]Leu-NH₂.

Unlike previously studied SP analogs the peptides of the invention arespecific to the SP receptor.Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ --NH]Leu-NH₂ was tested forthe ability to inhibit amylase release produced by various pancreaticsecretagogues (Table 4). Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂--NH]Leu-NH₂ (20 μM) inhibited amylase release stimulated by SP, but didnot alter bombesin, CCK-8, carbachol, VIP, secretin, CGRP A23187 or TPAstimulated amylase release.

The most potent receptor antagonists were tested for their ability toinhibit the binding of ¹²⁵ I-[Tyr⁴ ] bombesin to the bombesin receptorsof pancreatic acini. Spantide inhibited binding of ¹²⁵ I-[Tyr⁴ ]bombesin as reported previously (Jensen et al. (1988) Am. J. Physiol.254:G883) causing half-maximal inhibition at 3±1 μM and completeinhibition at 100 μM (FIG. 4).D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-LeuΨ[CH₂ --NH_(]) Nle-NH₂inhibited 125I-[Tyr⁴ ] bombesin binding but was 3-fold less potent thanspantide causing half-maximal inhibition at 10 μM (p<0.05 compared tospantide) (FIG. 4). Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂--NH]Leu-NH₂ did not cause detectable inhibition until concentrationsabove 30 μM and had a calculated K_(i) of 300±20 μM. Spantide andH-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-LeuΨ[CH₂ --NH]Nle-NH₂ had a3- to 10-fold lower affinity for inhibiting ¹²⁵ I-[Tyr]bombesin bindingas compared to .sup. 125 I-BH-SP. The ability ofH-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ --NH]Leu-NH₂ to inhibitbinding of ¹²⁵ I-[Tyr⁴ ] bombesin was 70-fold lower than its ability toinhibit binding of ¹²⁵ I-BH-SP.

Use

The peptides of the invention may be administered to a mammal,particularly a human, in one of the traditional modes (e.g., orally,parenterally, transdermally, or transmucosally), in a sustained releaseformulation using a biodegradable biocompatible polymer, or by on-sitedelivery using micelles, gels and liposomes.

The peptides can be administered to a human patient in a dosage of 0.5μg/kg/day to 5 mg/kg/day.

                  TABLE 1                                                         ______________________________________                                        The five carboxyl-terminal residues of a variety                              of bioactive peptides.                                                        Peptide        Carboxyl Terminal Sequence                                     ______________________________________                                        Bombesin       --Val--Gly--His--Leu--Met--NH.sub.2                            Neuromedin-β                                                                            --Thr--Gly--His--Phe--Met--NH.sub.2                            Neuromedin-C   --Val--Gly--His--Leu--Met--NH.sub.2                            Litorin        --Val--Gly--His--Phe--Met--NH.sub.2                            Neurokinin-A   --Phe--Val--Gly--Leu--Leu--NH.sub.2                            Neurokinin-B   --Phe--Val--Gly--Leu--Met--NH.sub.2                            Substance P    --Phe--Phe--Gly--Leu--Met--NH.sub.2                            Prototypical Tachykinin                                                                      --Phe--X--Gly--Leu--Met--NH.sub.2                              Sequence       (where X = a branched aliphatic or                                            aromatic amino acid residue)                                   ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Effect of the various SP and spantide derived pseudopeptides                  on basal and SP-stimulated amylase release.                                                               Amylase Release                                                               (percent total)                                   Peptide Added               Alone  SP (1 nM)                                  __________________________________________________________________________    None                        3.4 ± 0.5                                                                         6.6 ± 0.7                               H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--                                                                    3.1 ± 0.7                                                                         4.3 ± 0.5*                              Phe--Gly--LeuΨ[CH.sub.2 --NH]Leu--NH.sub.2 (10 μM)                     H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--                                                                     10.4 ± 1.0**                                                                     NT-agonist                                 Phe--GlyΨ[CH.sub.2 --NH]Leu--Leu--NH.sub.2 (10 μM)                     H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--PheΨ                                                            3.1 ± 1.0                                                                         5.4 ± 0.7*                              [CH.sub.2 --NH]Gly--Leu--Leu--NH.sub.2 (10 μM)                             H--Arg--Pro--Lys--Pro--Gln--Gln--                                                                         3.8 ± 0.7                                                                         6.4 ± 0.3                               PheΨ[CH.sub.2 --NH]Phe--Gly--Leu--Leu--NH.sub.2 (10 μM)                H--Arg--Pro--Lys--Pro--Gln--GlnΨ[CH.sub.2 --NH]                                                       3.1 ± 0.9                                                                         6.1 ± 1.0*                              Phe--Phe--Gly-- Leu--Leu--NH.sub.2 (10 μM)                                 H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--                                                                    3.8 ± 0.05                                                                       3.6 ± 0.1*                              Trp--Phe--D--Trp--LeuΨ[CH.sub.2 --NH]Nle--NH.sub.2 (10 μM)             H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--Trp--                                                              3.9 ± 0.8                                                                         4.2 ± 0.2*                              Phe--D--TrpΨ[CH.sub.2 --NH]Leu--Nle--NH.sub.2 (10 μM)                  H--D--Arg.Pro--Lys--Pro--Gln--Gln--D--Trp--                                                               4.0 ± 0.5                                                                         5.6 ± 0.8*                              PheΨ[CH.sub.2 --NH]D--Trp--Leu--Nle--NH.sub.2 (10 μM)                  H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--TrpΨ                                                           4.1 ± 0.5                                                                         4.8 ± 1.2*                              [CH.sub.2 --NH]Phe--D--Trp--Leu--Nle--NH.sub.2 (10 μM)                     H--D--Arg--Pro--Lys--Pro--Gln--GlnΨ[CH.sub.2 --NH]                                                    3.8 ± 0.3                                                                          4.5 ± 0.8**                            D--Trp--Phe--D--Trp--Leu--Nle--NH.sub.2 (10 μM)                            __________________________________________________________________________     *Significantly less than SP alone p < 0.05                                    **Significantly greater than no additions p < 0.01                            Acini were incubated at 37° C. for 30 min with 1 nM SP and 10 μ     concentrations of the various SP and spantide pseudopeptide analogues         either alone or in combination. Amylase release was expressed as percent      of amylase activity in acini at the start of incubation that was released     into extracellular medium during incubation. Values are means ± 1 SEM      from at least 5 separated experiments. In each experiment, each value was     determined in duplicate. Abbreviations: NTagonist = Not tested as an          antagonist because it was an agonist.                                    

                                      TABLE 3                                     __________________________________________________________________________    Abilities of SP, spantide and pseudopeptide to inhibit binding                of .sup.125 I-BH-SP or SP-stimulated amylase release.                                                           Inhibition of                                                         .sup.125 I-BH-SP                                                                      1nMSP-stimulated                                                      Binding Amylase release                             Peptide                   K.sub.i or K.sub.d (μM)                                                            IC.sub.50 (μM)                           __________________________________________________________________________    H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--Trp--                                                            2.1 ± 0.6                                                                          1.8 ± 0.1                                Phe--D--Trp--Leu--Leu--NH.sub.2 (Spantide)                                    H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--Trp--                                                            2.2 ± 0.4                                                                           1.8 ± 0.25                              Phe--D--Trp--LeuΨ[CH.sub.2 NH]Nle--NH.sub.2                               H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--Trp--                                                            3.6 ± 0.7                                                                          3.5 ± 0.6                                Phe--D--TrpΨ[CH.sub.2 --NH]Leu--Nle--NH.sub.2                             H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--Trp--                                                            14.7 ± 2.0                                                                          30 ± 5.0                                PheΨ[CH.sub.2 --NH]D--Trp--Leu--Nle--NH.sub.2                             H--D--Arg--Pro--Lys--Pro--Gln--Gln--D--TrpΨ                                                         6.3 ± 3.3                                                                          6.4 ± 1.4                                [ CH.sub.2 --NH]Phe--D--Trp--Leu--Nle--NH.sub.2                               H--D--Arg--Pro--Lys--Pro--Gln--GlnΨ                                                                 4.3 ± 1.1                                                                          4.7 ± 1.3                                [CH.sub.2 --NH]D--Trp--Phe--D--Trp--Leu--Nle-- NH.sub.2                       H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--                                                                  .0025 ± .0005                                                                      No-Agonist                                  Phe--Gly--Leu--Met--NH.sub.2 (SP)                                             H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--                                                                  4.3 ± 0.3                                                                           7.1 ± 0.9*                              Phe--Gly--LeuΨ[CH.sub.2 --NH]Leu--NH.sub.2                                H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--                                                                  5.6 ± 2.2                                                                          No-Agonist                                  Phe--GlyΨ[CH.sub.2 --NH]Leu--Leu--NH.sub.2                                H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--                                                                  41.3 ± 16.7                                                                        >30                                         PheΨ[CH.sub.2 --NH]Gly--Leu--Leu--NH.sub.2                                H--Arg--Pro--Lys--Pro--Gln--Gln--PheΨ                                                               265.0 ± 89.0                                                                       >30                                         [CH.sub.2 --NH]Phe--Gly--Leu--Leu--NH.sub.2                                   H--Arg--Pro--Lys--Pro--Gln--GlnΨ[CH.sub.2 --                                                        310.0 ± 88.0                                                                       >30                                         NH]Phe--Phe--Gly--Leu--Leu--NH.sub.2                                          __________________________________________________________________________     Values are means ± 1 SEM. Kd values for SP are obtained from Scatchard     analysis of .sup.125 Ilabeled SP binding studies K.sub.i values for           agonist or antagonists from studies of binding .sup.125 IBH-SP were           obtained according to the equation: K.sub.i = (R/1 - R) (SB/S + A) where      is the observed saturable binding of .sup.125 IBH-SP in the presence of       antagonist (B) expressed as a fraction of that obtained when B is not         present; A is the concentration of .sup.125 IBH-SP (0.125 nM). B is the       concentration of antagonist, S is the K.sub.d of SP determined by             Scatchard analysis. Noagonist = peptide not tested for inhibition activit     because agonist activity when present alone.                             

                  TABLE 4                                                         ______________________________________                                        Ability of                                                                    H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--Phe--Gly--                              LeuΨ[CH.sub.2 --NH]Leu--NH.sub.2 to affect amylase release stimulated     by                                                                            various secretagogues.                                                                     Amylase Release                                                               (percent total)                                                                           Leu.sup.11, Ψ10-11-SP                            Secretagogue   Alone     (20 μM)                                           ______________________________________                                        None            2.7 ± 0.4                                                                            2.3 ± 0.3                                        Substance P (1 nM)                                                                            8.7 ± 1.8                                                                            4.3 ± 0.5*                                       CCK-8 (0.1 nM) 19.7 ± 4.2                                                                           21.6 ± 4.9                                        Bombesin (0.3 nM)                                                                            14.8 ± 4.1                                                                           14.3 ± 3.1                                        Carbachol (10 μM)                                                                         22.7 ± 4.1                                                                           20.7 ± 4.3                                        VIP (0.3 nM)   20.5 ± 3.5                                                                           18.9 ± 4.2                                        Secretin (0.1 μM)                                                                         18.9 ± 3.6                                                                           18.8 ± 4.1                                        CGRP (0.1 μM)                                                                             12.1 ± 3.3                                                                           12.0 ± 3.9                                        A23187 (0.1 μM)                                                                            9.9 ± 1.5                                                                           11.6 ± 1.6                                        TPA (0.1 μM)                                                                              32.2 ± 5.7                                                                           30.2 ± 5.6                                        ______________________________________                                         *Significantly less than secretagogue alone p < 0.001                         Acini were incubated for 30 min at 37° C. with various pancreatic      secretagogues alone or with                                                   H--Arg--Pro--Lys--Pro--Gln--Gln--Phe--Phe--Gly--LeuΨ[CH.sub.2             --NH]LeuNH.sub.2. In each experiment, each value was determined in            duplicate, and results give means ± 1 SEM from at least 4 separated        experiments. Abbreviations: CCK8, COOHterminal octapeptide of                 cholecystokinin; VIP, vasoactive intestinal peptide, respectively; CGRP,      calcitonin generelated peptide; A23187 and TPA,                               1,2o-tetradecanoylphorbol-1,3-acetate.                                   

Other embodiments are also within the claims set forth below.

What is claimed is:
 1. A substance P analog of the formula: ##STR10##wherein A¹ =the D or L isomer of Arg, Lys, or Lys-ε-NH-R₂₀ in which R₂₀is any of H, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, or COE₁₀ with E₁₀ beingC₁₋₂₀ alkyl, C₃₋₂₀ alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀phenylalkyl; or is deleted;A² =the D or L isomer Pro; or is deleted; A³=the D or L isomer of Lys, or Lys-ε-NH-R₂₂ in which R₂₂ is any of H,C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, or COE₁₂ with E₁₂ being C₁₋₂₀ alkyl,C₃₋₂₀ alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl; oris deleted; A⁴ =the D or L isomer of Pro; or is deleted; A⁵ =the D or Lisomer of Asp, Gln, β-Nal, Trp, Phe, o-X-Phe in which X is F, C1, Br,NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ;or is deleted; A⁶ =the D or L isomer of Ala, Arg, Ser, Pro, Gln, p-Glu,Asn, β-Nal, Trp, Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃,or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ; A⁷ =the D or Lisomer of Val, Thr, Phe, Trp, β-Nal, o-X-Phe in which X is F, Cl, Br,NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ;A⁸ =the identifying group of Gly or the D or L isomer of Val, Trp,β-Nal, Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phein which X is F, Cl, Br, NO₂, OH, or CH₃ ; A⁹ =Gly or the D or L isomerof Sar, His, Trp, β-Nal, Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH,or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ; A10=Gly orthe identifying group of the D or L isomer of Trp, β-Nal, Leu, Nle, Ala,cyclohexyl-Ala, Val, Ile, Met, Phe, o-X-Phe in which X is, Cl, Br, NO₂,OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ;A11=Gly or the identifying group of the D or L isomer of Trp, β-Nal,Leu, Nle, Ala, Val, Ile, Met, Phe, o-X-Phe in which X is F, Cl, Br, NO₂,OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ; or isdeleted;R₅ is CH₂ --NH and V¹ is ##STR11## in which each R₁₀, R₁₁, andR₁₂ independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, or C₁₂₋₂₀naphthylalkyl; further provided that, where A⁵ is Asp, A⁶ is Ser, A⁷ isPhe, A⁸ is Val, A⁹ is Gly, A¹⁰ is Leu, and A¹¹ is Leu, at least one ofA¹, A², A³, or A⁴ must be present; or a pharmaceutically acceptable saltthereof.
 2. A substance P analog of the formula: ##STR12## wherein A²¹=the D or L isomer of Arg, Lys, or Lys-ε-NH-R80 in which R₈₀ is any ofH, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, or COE₂₀ with E₂₀ being C₁₋₂₀ alkyl,C₃₋₂₀ alkenyl, C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl; oris deleted;A²² =the D or L isomer of Pro; or is deleted; A²³ =the D or Lisomer of Lys or Lys-ε-NH-R₈₂ in which R₈₂ is any of H, C₁₋₁₂ alkyl,C₇₋₁₀ phenylalkyl, or COE₂₂ with E₂₂ being C₁₋₂₀ alkyl, C₃₋₂₀ alkenyl,C₃₋₂₀ alkynyl, phenyl, naphthyl, or C₇₋₁₀ phenylalkyl; or is deleted;A²⁴ =the D or L isomer of Pro; or is deleted; A²⁵ =the identifying groupof the D or L isomer of Asp, Gln, B-Nal, Trp, Phe, o-X-Phe in which X isF, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe in which X is F, Cl, Br, NO₂, OH,or CH₃ ; or is deleted together with NH--CH bonded thereto; A²⁶ =theidentifying group of the D or L isomer of Arg, Sar, Pro, Gln, pGlu, Phe,Trp, cyclohexyl-Ala, or Asn; A27=the identifying group of D-Trp; or theidentifying group of the D or L isomer of Leu, Phe, or cyclohexyl-Ala;A²⁸ =the identifying group of the D or L isomer of any one of the aminoacids Val, β-Nal, Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃,or p-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ; A²⁹ =theidentifying group of the amino acid D-Trp; or the identifying group ofthe D or L isomer of any of Leu, Phe, or cyclohexyl-Ala; A³⁰ =theidentifying group of the D or L isomer of any one of the amino acidsLeu, Nle, Ala, cyclohexyl-Ala, Val, Ile, Met, Gly, Phe, Trp, β-Nal,o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃, or p-X-Phe in which Xis F, Cl, Br, NO₂, OH, or CH₃ ; A³¹ =the identifying group of the D or Lisomer of any one of the amino acids Trp, β-Nal, Leu, Nle, Ala, Val,Ile, Met, Gly, Phe, o-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃, orp-X-Phe in which X is F, Cl, Br, NO₂, OH, or CH₃ ;R₅₄ is CO--NH orCO--NCH₃ if A²⁵ with NH--CH is not deleted, is NH if A²⁵ with NH--CH isdeleted; each R₅₆, R₆₂, and R₆₄, independently, is any of CO--NH or CH₂--NH; R₅₈ is CO--NR₆₉ in which R₆₉ is H or C₁₋₁₂ alkyl, or CH₂ --NH; R₆₀is CO--NH; and V¹⁰ is ##STR13## in which each R₆₆, R₆₇, and R₆₈independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀ phenylalkyl, or C₁₂₋₂₀naphthylalkyl; provided that, at least one of R₅₆, R₅₈, R₆₂, or R₆₄ isCH₂ NH; or a pharmaceutically acceptable salt thereof.
 3. The substanceP analog of claim 1 of theformula:H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-LeuΨ[CH₂ --NH]Leu-NH₂ ; ora pharmaceutically acceptable salt thereof.
 4. The substance P analogsof claim 2 of theformula:H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-TrpΨ[CH₂--NH]Leu-Nle-NH₂ ; or a pharmaceutically acceptable salt thereof.
 5. Thesubstance P analogs of claim 2 of theformula:H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Ψ[CH₂--NH]Nle-NH₂ ; or a pharmaceutically acceptable salt thereof.
 6. Thesubstance P analogs of claim 2 of theformula:H-D-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Ψ[CH₂--NH]Phe-D-Trp-Leu-Nle-NH₂ ; or a pharmaceutically acceptable saltthereof.
 7. The substance P analogs of claim 2 of theformula:H-D-Arg-Pro-Lys-Pro-Gln-Gln-Ψ[CH₂--NH]D-Trp-Phe-D-Trp-Leu-Nle-NH₂ ; or a pharmaceutically acceptable saltthereof.
 8. The substance P analog of claim 1 in which each A¹ throughA⁵ is an amino acid.
 9. The substance P analog of claim 2 in which eachA²¹ through A²⁵ is an amino acid.